This proposal's long term goal is to determine the molecular and developmental mechanisms by which Eya and its cofactors Six act during mammalian inner ear development. The vertebrate inner ear develops from the otic placode via multiple inductive processes. A large number of otic genes have been isolated recently;however, their precise functions in inner ear and its sensory cell development are largely unknown. Eya1 has been shown to be a key gene for inner ear development: mutations in the human EYA1 gene cause Branchio-Oto-Renal (BOR) syndrome, a congenital birth defect that accounts for as many as 2% of profoundly deaf children, while inactivation of Eya1 in the mouse causes an arrest of inner ear development at the otocyst stage. However, despite the identification of the responsible gene for BOR syndrome, the developmental and molecular basis of auditory defects occurring in BOR syndrome and the identity of the steps at which Eya1 functions in inner ear development are unclear. During the current grant period, we investigated the developmental and molecular basis of inner ear defects associated with Eyaf-deficiency. We identified mutations in the human SIX1 or SIX5 from BOR patients and analyzed these mutations functionally, identified the developmental functions of Six1 in the auditory system through gene targeting, tested the interactions between Six1 and Eya1 in vivo, and clarified the regulatory relationship between Pax, Eya and Six genes. In addition, we have identified Six1 regulatory elements that direct expression in specific parts of the developing inner ear. Furthermore, we generated Eya2 mutant mice, and the homozygous mice have hearing loss. This renewal application will continue to define the molecular and developmental mechanisms by which Eya and Six genes act during inner ear development. Specifically, we propose: (1) To determine whether Eya and Six genes play a crucial role in determination and development of the otic placodes, (2) To identify regulatory elements controlling Six1 otic expression and rigorously evaluate its regulation by Eya1 via genetic and molecular approaches, (3) To define the biological function of Eya2 during inner ear development, and (4) To further determine the role of Eya1, Six1 and S/x5-three BOR syndrome causing genes-in inner ear development. These studies should yield important new information about developmental and molecular mechanisms of inner ear morphogenesis, and greatly extend our understanding of the roles of Eya and its cofactors Six in otic placode induction and sensory cell development in the mammalian inner ear. The results will provide important insights into the developmental and molecular pathogenesis of inner ear defects in the BOR syndrome.